The overall goal of this proposal is to define the mechanism of cyclic AMP (cAMP) signaling pathways in the liver and pancreatic 2-cell. Elevated intracellular cAMP in the hepatocyte increases hepatic glucose production, while the same signal in the pancreatic 2-cell promotes insulin secretion and proliferation. The cAMP signal activates protein kinase A (PKA), which phosphorylates the nuclear cAMP response element binding protein (CREB). Phosphorylated CREB recruits the nuclear co-activators CREB binding protein (CBP), and the related protein p300. CBP, but not p300, contains an insulin phosphorylation site at serine 436. Insulin phosphorylation at serine 436 is required for efficient dissociation of the CREB-CBP complex and consequent attenuation of CREB-dependent gene transcription. We have generated a knock-in mouse model where CBP is mutated at serine 436 (CBP-S436A), removing this site of insulin phosphorylation. As a result, CREB-dependent gene transcription is increased and resistant to the inhibitory nuclear effects of insulin. These mice show enhanced hepatic glucose production and pancreatic 2-cell hyperplasia associated with a defect in glucose-stimulated insulin secretion. These effects may be mediated by the PPAR3 co-activator 11(PGC-1 1, a cAMP target gene) whose expression is increased in both the hepatocyte and pancreatic 2-cell of these animals. PGC-11 activates a fasting gene program in liver and impairs energy production in the 2-cell. In this proposal, we will attempt to understand if PGC-1 1 mediates the CBP S436A mouse phenotype, since this mutation may affect other signaling pathways (Aim 1). CBP and p300 may have discrete physiological roles based on the presence or absence, respectively, of an insulin phosphorylation site. Aim 2 will establish the relative importance and mechanism of hepatic and 2-cell regulation mediated by these closely related co- activators by studying a mouse model where an artificial insulin phosphorylation site is introduced in p300 (G421S). Finally, CBP remains in the nucleus regardless of its phosphorylation state, while the forkhead protein, FoxO1, is excluded from the nucleus after insulin phosphorylation. FoxO1 has been reported to play a dominant role in activating hepatic gluconeogenesis and inhibiting 2-cell growth based on over-expression mouse models. Aim 3 will compare the effects of insulin phosphorylation mutants of CBP and FoxO1, expressed at allelic levels, on hepatic glucose production and 2-cell regulation and/or growth. These studies are significant because they will establish signaling pathways that normally regulate the hepatocyte and pancreatic 2-cell in vivo and provide a mechanistic understanding of pathophysiological changes found in patients with diabetes mellitus. PUBLIC HEALTH RELEVANCE: In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic 2-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements.